![]() ![]() Adapted from Lakowicz.² The Appearance of Second Order Diffraction in Fluorescence Spectraįigure 2: Example of second order artefacts in a broad fluorescence emission spectrum of a solution of 2-aminopyridine mixed with Ludox excited at 300 nm. The consequence of this is that when the monochromator is set to transmit 600 nm, a small fraction of 300 nm light will also be transmitted which can be problematic for fluorescence spectroscopy.įigure 1: The overlapping orders of a diffraction grating. Consider light at 600 nm that is first order diffracted (m = 1, λ = 600 nm) and light at 300 nm that is second order diffracted (m = 2, λ = 300) it is clear that the left hand side of the grating equation is the same for both cases and the angle of the diffracted light must therefore be equivalent. This shared range can also be seen from the grating equation. This is illustrated in Figure 1 where the blue cone represents the range of angles where the light is first order diffracted and the red cone is the range of angles where the light has been diffracted second order and there is an overlap region shared between these ranges. However, due to the broad range of wavelengths being diffracted, the angle ranges occupied by the first and second order diffraction are not unique. ![]() In a monochromator it is only the first order diffraction (either +1 or -1) that is used to select the desired wavelength and the higher orders are unwanted. Diffraction at higher orders follows a similar pattern of increasing angle away from the normal and reducing intensity. Similarly a value ☒ is known as second order diffraction and occurs at a shallower angle and is weaker in intensity. A value of ☑ is termed first order diffraction and occurs closet to the grating normal and is the highest in intensity. It can also be seen that for constant and constant λ the equation is satisfied with different angles depending on the diffraction order m which can take positive and negative integer values (…-2, -1, 0, 1, 2…). It can be seen that for constant each wavelength of light will be diffracted at a different angle which allows the monochromator to isolate the desired wavelength. Where m is the order of the diffraction, λ is the wavelength of the diffracted light, d is the groove spacing of the grating, is the angle between the incident light and the grating normal, θ ί is the angle between the diffracted light and the grating normal. The broadband light is shone on the diffraction grating and the different wavelengths that comprise the light are diffracted at different angles in order to satisfy the grating equation, ![]() Monochromators utilise diffraction gratings in order to isolate the desired wavelength from incident broadband light. A typical fluorescence spectrometer will consist of two monochromators an excitation monochromator to select the desired excitation wavelength and an emission monochromator to select which wavelength reaches the detector. In fluorescence spectroscopy, monochromators are used to select the excitation and emission wavelengths. In this Spectral School tutorial we discuss the phenomena of second order diffraction through a monochromator and the problems it can cause in fluorescence spectroscopy.
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